Drug addiction is a devastating disease affecting millions of people in the United States. At the core of this behavior are complex biochemical processes that affect individual neurons and neuronal circuits. A major challenge is to understand the molecular and cellular basis of addiction and associated behaviors of dependence, withdrawal, tolerance, and sensitization. Many abused drugs act directly or indirectly on receptors that in turn activate the Gi/Go class of heterotrimeric G proteins and upon chronic stimulation of these receptors, the cAMP signaling pathway is up-regulated. An early adaptive response to chronic Gi stimulation is an increase in the activity of adenylyl cyclase termed adenylyl cyclase super-sensitization. Studies over the years have provided a great deal of information regarding the molecular basis responsible for adenylyl cyclase super-sensitization and have implicated the involvement of many of the regulators of adenylyl cyclase, including Gs1, Gi1, 23, and protein kinases;however, the major mechanisms underlying this phenomenon is still largely unknown. My laboratory has developed a cell based model system for of adenylyl cyclase super-sensitization that is amenable to moderate high throughput screening approaches. This work has allowed us to develop and execute a genome wide siRNA screen to identify genes that when silenced, block the adenylyl cyclase super-sensitization that develops in response to chronic hormonal activation of a Gi pathway. We will examine the phosphorylation of adenylyl cyclase that occurs during adenylyl cyclase super- sensitization (aim 1), study the role of TTK, a dual-specificity protein kinase protein that we identified in our screen in the development of the super-sensitivity of the adenylyl cyclase enzyme (aim 2). Our results from the siRNA screen, as well as follow up studies implicate a role of protein turnover in the development of adenylyl cyclase super-sensitization;aim 3 of the proposal examines the role of the ubiquitin-mediated protein degradation pathway in adenylyl cyclase super-sensitization. In aim 4 we will characterize the role of an identified AKAP target of the ubiquitin-mediated protein turnover machinery in the development of adenylyl cyclase super-sensitization. Our genome-wide siRNA screen has produced a rich dataset that will provide us with a unique opportunity to identify novel components of the signaling pathways that underlie adenylyl cyclase super-sensitization. This will provide insights into the biologically important process of drug addiction.